Enteric diseases caused by enterotoxigenic E. coli (ETEC) strains, Shigella spp, and Salmonella Typhi, all of which are NIAID Category B priority agents, collectively affect > 400 million people annually worldwide. Currently, there is no vaccine against ETEC or shigellosis. A typhoid vaccine exists. An affordable, effective, oral multivalent vaccine against all 3 organisms would have enormous public health importance, and a substantial commercial market among travelers and military personnel. Our long-term goal is to create a stable, orally administered vaccine against ETEC, shigellosis and typhoid. To begin the process of achieving this goal we have used the licensed Ty21a typhoid vaccine to express the O-antigen of Shigella sonnei to produce a vaccine candidate called TyOraSs, which is under development. The major virulence determinants of ETEC are the colonization factor antigens (CFAs or adhesins) and two enterotoxins, the heat-labile (LT) and heat-stable toxin (STa). An effective ETEC vaccine should induce antibodies that block bacterial attachment and/or to neutralize the toxins. In animal models antibodies to CFA and toxin are synergistically protective. It has been shown by members of our team that a multi-epitope fusion antigen (MEFA) representing 7 separate CFA ETEC adhesins and two toxins can be fused as a single protein (designated here as MEFA+T) to induce cross-protective antibodies that blocks adherence of heterogeneous ETEC strains to human colon cancer cells in vitro, and neutralizes two toxins in all ETEC strains. In this project we will stably express these multiple adhesins and the toxoid form of both toxins stably as a holotoxin structured CFA-toxoid fusion cassette antigen in Ty21a, and assess immunogenicity and protective efficacy of our Ty21a-ETEC vaccine using the suckling piglet and rabbit challenge models. Specifically we will, 1) Generate and characterize vaccine strain(s) of genetically optimized Ty21a expressing chromosomally integrated, ETEC multi-epitope fusion antigen (MEFA) + toxoid LT-STa (designated Ty21a-ETEC MEFA-T) either intra-cellularly or in secreted form, 2) Demonstrate immunogenicity against ETEC and S. Typhi, by mucosal immunization of mice, and 3) Establish protective efficacy against ETEC in the rabbit colonization model and suckling piglet lethal infection model. Our proposal is unique because of our expertise at construction of multivalent ETEC fusion antigens, experience with using Ty21a as a platform for expressing heterologous antigens, and capabilities with animal models to unambiguously assess vaccine protective efficacy. A stand-alone ETEC-typhoid vaccine would have substantial impact, however our aim to use success in this project as a foundation for the development of a multivalent vaccine against ETEC, typhoid, and shigellosis. In Phase II we will generate a single triple pathogen vaccine, TyOraSs-ETEC vaccine, or two bivalent vaccines; generate a master cell bank of the vaccine candidate strain(s) for manufacturing in compliance with cGMPs and as a foam-dried vaccine product(s); conduct required pre-clinical studies; design a clinical protocol; and prepare an IND.